Abstract

In 2021, coronary artery disease (CAD) was responsible for 375,476 fatalities. Many of these deaths could be prevented by improved diagnosis, cost-effectiveness, and less invasive procedures. Through CADlock, we promoted early diagnostics and inclusive care for underrepresented demographics. We met this goal by emphasizing accessibility, affordability, and accuracy through procedures providing preliminary care to systematically marginalized groups before disease progression. By adding a nicking endonuclease, we enhanced rolling circle amplification (RCA) into exponential RCA, improving reaction accuracy. Additionally, we improved Micro-Q by utilizing a white LED to test various fluorescence wavelengths in a single frugal device. To further accommodate the accessibility of miRNA research, we created CADmir: a comprehensive search engine utilizing large language models to organize miRNA information. Finally, we educated our peers and teachers on the applications of biotechnology, inspiring future generations to make healthy, proactive lifestyle choices.

Defining the problem

Coronary artery disease (CAD) currently ranks as the third highest cause of death worldwide. CAD was responsible for 375,476 fatalities (CDC, 2019). It is also the number one cause of death in the United States, specifically in the southeastern region including our home state: Georgia (CDC, 2019) (see Fig. 1).

CAD exhibits various symptoms, some of which cannot be visible (CDC, 2019). Moreover, women typically show symptoms 7 to 10 years later than men due to the high levels of estrogen in their bodies premenopause (Brown, 2022). The following displays various common symptoms linked to CAD (Maas & Appelman, 2010).

• Chest pain (angina), fullness or pressure
• Fatigue
• Heart palpitations, or sensations of a racing heartbeat
• Shortness of breath (dyspnea)
• Swelling in the hands or feet

Currently, various detection and diagnosis methods are used for CAD. These include angiograms, computed tomography angiogram scans, echocardiograms, electrocardiograms, and nuclear stress tests (Nuclear Cardiac Stress Test: What to Expect, n.d., 2022). These tests, however, are not affordable, and some are highly invasive. Moreover, there are certain harmful side effects of certain stress tests, such as chest pain, which can increase a patient’s risk for a sudden heart attack ( Nuclear Cardiac Stress Test: What to Expect, n.d., 2022). To mitigate this, Lambert iGEM 2023 further emphasizes the importance of inclusivity by pursuing a more accurate and precise detection mechanism by broadening the scope of accessibility.

Figure 1. Map of coronary artery disease distribution in the United States.

Pathophysiology

Coronary artery disease is caused by plaque buildup which is initially clustered in fat and blood cells in the body (see Fig. 2). As the collection worsens, arteries narrow in a process called atherosclerosis. The following describes the key process in three crucial steps from the normal condition of the artery (Shahjehan, 2023).

Figure 2. The progression of CAD

MicroRNA/2022 Project

MicroRNAs (miRNAs) are a class of non-coding, port-transcriptional RNA molecules that play essential roles in regulating gene expression. miRNAs are about 20-23 nucleotides in length, and they enhance or inhibit protein production via translational repression, or the cleaving of mRNA sequences. miRNAs are first transcribed from DNA sequences into primary miRNAs, then processed into precursor miRNAs, and finally transcripted into mature miRNAs (see Fig. 3). Most miRNAs are identified by their 5p position (forward 5’ - 3’) and their 3p position reverse (3’ - 5’). miRNAs interact with target mRNAs with 3′untranslated regions (3′ UTR) to induce cleavage and translational repression (O’Brien et al., 2018). However, miRNAs also interact with other regions, including the 5′ UTR, coding sequences, and gene promoters (NIH, 2022).

Figure 3. Mature miRNA editing

In 2022, we tested miRNA hsa-miR-1-3p, which is upregulated in coronary artery disease (CAD), as a biomarker for screening the disease. This means it can detect the miRNAs at necessary concentrations in conjunction with padlock probes, which are long strands of DNA with ends (or arms) complementary to the target miRNA (See Fig. 4). By utilizing a process known as rolling circle amplification (RCA), we amplified the padlock sequence creating a product up to 10 kilobases long. Then, using the reporting mechanism called linear DNA probes, we successfully measured the fluorescence output with Micro-Q, our frugal fluorometer.

Figure 4. Image description of hybridization of miRNA and padlock probes

Barriers

Lambert iGEM had certain concerns in their 2022 project to which solutions and alternatives are being approached and pursued in 2023. The 2022 project held concerns about the lack of accuracy with the padlock probes and microRNAs (miRNAs) and inefficient performance from the aptamers. Taking these factors into consideration, the team has developed a second phase of CADLOCK which consists of improved wetlab and hardware aspects that increase the efficiency of proactive care through innovations with accessibility, accuracy, and affordability.

Capillary tube: To improve the point of care and accessibility aspects of the 2022 project, the team began the development of capillary RCA, which negates the need for a fluorometer by using capillary tubes for visual, counting-based quantification. By removing the necessity of a fluorometer and expensive diagnostic equipment, capillary RCA could potentially enable more accessible and cost-effective testing of CAD.

MircroQ: After extensive in-field testing in Thailand, researchers determined two main issues to address while developing our new iteration of Micro-Q: its inability to fluoresce multiple samples simultaneously and fluorescing samples at different wavelengths. With this in mind, the team developed Micro-Q Pro, a device capable of exciting 8 PCR tubes containing samples that fluoresce anywhere from 400 to 700 nm. Utilizing a white LED strip, a 3D printed holster, and an ESP-32 Camera we were able to accurately detect and quantify fluorescence.

Exponential RCA: Although RCA is highly sensitive to the target miRNAs, it cannot differentiate between various concentrations of miRNA. To address this, the Lambert iGEM team is pursuing exponential RCA (eRCA), an adaptation of the rolling circle amplification model that produces exponentially greater fluorescence per unit of miRNA, therefore increasing between miRNA concentration and margin of error.

Estrogen: Females are more susceptible to CAD due to their fluctuating levels of estrogen. Essentially, estrogen acts as a CAD suppressor, and so when females undergo menopause with a drastic estrogen drop, they have an increased risk of developing CAD. Lambert iGEM expanded its target population to men as well. Since men have stable levels of estrogen, they would be an accurate population to test for consistent results. Lambert iGEM experimented with mir-20-b which, with a high level in men, indicates CAD, while in premenopausal women, indicates protection from CAD.

Protein Purification: While RCA provides a more frugal alternative to existing methods, costing $8.98, phi2DNA polymerase makes up approximately 56% of the cost. In addition to this, multiple RCA reactions needed to be performed in order for an accurate diagnosis, increasing the total cost significantly. To increase the preventative aspect of healthcare and decrease the cost of the overall assay, we produced phi29 DNA polymerase through protein purification, a method to cheaply produce phi29.

CADmir: To address the need for a database that extracts functional miRNA information and organizes the vast volume of scientific literature regarding miRNAs, we developed CADmir: a miRNA search engine utilizing large language models to process and organize the extensive information about miRNAs. With CADmir, most of the relevant data about specific miRNAs are consolidated in one place, improving information accessibility and reducing the time barrier associated with obtaining high-quality scientific information. CADmir works alongside existing databases such as miRBase to provide links to relevant scientific literature and incorporate sequence and genomic data. CADmir expands research in miRNAs and creates a new precedent for the organization of vital scientific information.

Lab Pilot: Pipetting is a fundamental laboratory technique used to transfer precise volumes of liquid from one container to another. Consistent pipetting is essential for achieving accurate results, maintaining data integrity, and facilitating advancements in synthetic biology research. However, manual pipetting limits throughput and is easily susceptible to human error. To resolve these issues, automated liquid handlers are designed to automate and streamline the process of pipetting liquids in laboratory settings. However, current automated liquid handlers costing between $5000 and $300,000 are difficult to use. Because of this, the majority of current labs don’t have easy access to an automated liquid handler. To provide underfunded labs access to liquid handlers, Lambert iGEM developed LabPilot, a frugal liquid handler made with 3D printable and accessible parts that can accurately pipette based on direct user input. LabPilot is an automated liquid handler compatible with a variety of micropipettes. It utilizes an XYZ layout for movement, with most of its structural parts designed on Fusion 360, a computer-aided design application.

Our Approach

2023 Lambert iGEM has undertaken a new approach with the project’s primary focus area as inclusivity in order to enable 2023 CADlock to be readily available regardless of sex, ethnicity, or socioeconomic status. To address this initiative, Lambert iGEM implemented several tactics, including an exponential approach to improve the accuracy of RCA, a sex inclusive wetlab initiative, a cost-effective fluorometer that accommodates various wavelengths, a prototype for an automated pipetter, an accessible database for CAD microRNAs, a STEM inclusivity podcast, and a “Science for All” webinar. We spoke with multiple researchers and cardiologists who aided our decisions regarding the improvement of our hardware and wetlab systems. Our team builds upon CADlock by creating improved wetlab and frugal hardware devices and taking a holistically proactive approach through accuracy, affordability, and accessibility.

References